Titel deutsch:

Analyse der SDF‑1a Expression in hMSC für die experimentelle Untersuchung der Chemotaxis von hämatopoietischen Stammzellen

Titel englisch:

Analysis of SDF‑1a expression in hMSC for the experimental investigation of hematopoietic stem cell chemotaxis

Autoren:

Christina Leinweber

Rainer Saffrich

Anthony D. Ho

Michael Grunze

Axel Rosenhahn

Institutes:

Angewandte Physikalische Chemie, Universität Heidelberg

Innere Medizin V, Hämatologie und Onkologie, Universitätsklinikum Heidelberg

Institut für Funktionelle Grenzflächen (IFG), Karlsruhe Institute of Technology (KIT)

Introduction: Human mesenchymal stromal cells (hMSC) can be used as a niche model for the experimental investigation of the chemotactic migration of human hematopoietic stem and progenitor cells (HSC) towards their stem cell niche, the so called homing process. These bone marrow derived hMSC express the messenger chemokine stromal cell-derived factor‑1alpha (SDF‑1a). Because the exact sensing mechanism and migration behavior of HSC is still not completely unraveled, we studied migration of human HSC under laboratory conditions using several chemotaxis experiments with increasing intricacy from single well up to microfluidic devices. Methods: For the definition of chemical gradients inside these setups we explored in detail the expression of the chemokine SDF‑1a in hMSC applying ELISA tests (RayBiotech). The expression behavior has been studied dependent on culture time, cell amount and culture media. Additionally we performed intracellular immunofluorescence staining of SDF‑1a in hMSC to support the ELISA results. Human HSC (CD34+) for migration experiments were derived from cord blood or mobilized peripheral blood. Both, HSC and hMSC, as well as recombinant SDF‑1a were used to investigate HSC chemotaxis in microwell, transwell and microfluidic migration setups. Results: The media test clearly shows that SDF‑1a expression in hMSC is strongly dependent on culture conditions. Highest expression rates and values were obtained within a commercial MSC culture medium (Lonza). Therein SDF‑1a expression increases with increasing cell amount and longer culture time. About 24 hours after a medium exchange an equilibrium plateau concentration in the medium supernatant (about 200pg/ml for 50.000 cells) is reached in most cases. The chemotactic response of HSC correlates with the expected development of chemokine gradients according to the measured production rates and diffusion profiles inside the applied microwell and microfluidic setups. Conclusions: With our study we demonstrate the importance of hMSC culture conditions in planning in-vitro chemotaxis and homing experiments with hMSC as a niche model. SDF‑1a expression data measured with ELISA can be used to calculate chemical gradients inside of microfluidic setups. Thus it is possible to determine sensing thresholds and sensitivity of HSC in response to SDF‑1a. Furthermore we showed the applicability of a simple microfluidic setup in experimental medical research.

Disclosure: No conflict of interest disclosed.